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. 2019 Apr 12;11(4):525. doi: 10.3390/cancers11040525

Figure 6.

Figure 6

The carbonyl cyanide m-chlorophenylhydrazone (CCCP) exposure triggers reactive oxygen species (ROS) production, mitochondrial depolarization, and autophagy in T98 and U251 cells. (a) Lysates from T98 and U251 cells, untreated or treated for 24 h and 48 h with CCCP, were separated on 14% SDS-PAGE and probed with anti-LC3 and anti-GAPDH Abs. GAPDH protein levels were evaluated as loading control. Blots are representative of one of three separate experiments. Bars represent the densitometric analysis. * p < 0.05 vs. untreated cells. (b) PI incorporation was analyzed by flow cytometry in T98 and U251 cells treated as described above. Histograms are representative of one of three separate experiments. MFI = mean fluorescence intensity. (c) To analyze ROS production in GBM cells, treated as described above, were stained with dichlorodihydrofluorescein diacetate (DCFDA) before flow cytometric analysis. Histograms are representative of one of three separate experiments. (d) T98 and U251 cells were treated with CCCP as described above and the mitochondrial transmembrane potential (ΔΨm) changes were evaluated by tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining and biparametric FL-1(green)/FL-2(red) flow cytometric analysis. Data are representative of one out of three separate experiments.